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Chapter 5. Conclusion
5-1. Chapter summary
The macromolecular therapeutics have contributed to cancer treatment by improving therapeutic efficacy and reducing systemic side effects. These advantages of the macromolecular therapeutics are resulted in the spontaneous and selective accumulation in solid tumor by the enhance permeability and retention (EPR) effect. The EPR effect is caused by a wide gap in tumor vascular endothelia and undeveloped lymphatic vessel in the tumor tissue. However, the tumor accumulation by the EPR effect has been anticipated to be improved. The obstacles in the EPR effect are the limited blood flow, barrier of endothelial tight junction, abnormal extracellular matrix and interstitial and tumor growth-induced solid stress. To overcome these obstacles, the author utilized nitric oxide (NO), which is an endogenous vasodilator and a vascular permeability enhancer. To achieve a tumor-specific vasodilation, the author utilized macromolecules to specifically deliver NO donor to tumor by the EPR effect.
In chapter 2, the author developed a NO donor-containing PEGylated liposome (NONOate-LP). DETA-NONOate was encapsulated into the liposome during the extrusion procedure, which enabled extension of a half-life of NO release. The author found that NONOate-LP accumulated in solid tumors more than control groups (empty LP, empty LP + free NONOate). In addition, NONOate-LP did not show hepatotoxicity and nephrotoxicity. The author proposed that NONOate-LP enhanced tumor accumulation by positive feedback.
In chapter 3, the author examined further analysis of the accumulation profile of NO donor-containing liposome (NO-LP) to prove the positive feedback. The author found that NO-LP showed enhanced accumulation in the early stage (1 h) and the accumulation continued to increase until 48 h after the injection. In contrast, accumulation of control groups (empty LP, empty LP + free NONOate) became saturated 24 h after the injection.
This continuous accumulation of NO-LP strongly suggested the positive feedback in the enhancement of the EPR effect. The author also examined storage stability of NO-LP at 4 ºC and found that 80% of the NONOate still remained in NO-LP at day 25. NO-LP
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showed sustained NO release at physiological temperature. Moreover, the NO release speed of NO-LP was not affected by pH of outer solution. These are suitable characteristics of NO-LP for practical application.
In chapter 4, the author developed a mAb-NO donor conjugate to enhance the tumor accumulation of mAb by positive feedback mechanism to increase the therapeutic efficacy. The preparation procedures of cetuximab conjugated with S-nitrosothiol groups (Ab-SNO) were established. Ab-SNO released NO in physiological condition with a favorable speed, while it maintained original functions such as the binding ability to EGFR and effector function. In vivo study showed that Ab-SNO boosted tumor accumulation of Ab-Cy7 and Evans blue/serum albumin complex. Ab-SNO tended to suppress the tumor growth more efficiently than cetuximab.
5-2. Conclusion
In tumor environment, the limited blood flow suppresses the delivery of the drugs. By using NO, the EPR effect is expected to be enhanced by increasing blood flow in tumor and loosening of tight junction of endothelia of tumor vasculature. For the tumor specific induction of vasodilation, the author embedded NO donors on macromolecular therapeutics to release NO specifically in tumor by the EPR effect. First, the author developed NO donor-containing PEGylated liposomes (NONOate-LP and NO-LP).
These NO donor-containing liposomes successfully enhanced their own tumor accumulation and the tumor accumulation of co-administered liposomes which did not contain NO donor. The enhancement of the tumor accumulation of co-administered doxorubicin containing liposome (Dox-LP) tended to suppress tumor growth more efficiently than the group treated with Dox-LP alone. The NO donor-containing liposomes showed enhanced tumor accumulation rapidly (in 1 h) and continuously (for 48 h). From the observation, the author proposed the positive feedback mechanism works on the enhanced EPR effect of the NO donor-containing liposomes. Second, NO donor modification of macromolecular therapeutics was applied to mAb. The author established procedure to modify S-nitrosothiol groups on cetuximab. Ab-SNO thus obtained released
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NO in a favorable speed, while it maintained its original functions. Ab-SNO boosted the EPR effect of co-administered mAb and endogenous serum albumin/Evans blue complex.
Ab-SNO tended to suppress tumor growth more efficiently than cetuximab.
5-3. Perspective
In this thesis, the author developed NO donor-embedded macromolecular therapeutics (liposome and mAb) to enhance their own tumor accumulation and the accumulation of the co-administered macromolecular therapeutics. The author proposed that positive feedback mechanism works on the enhanced EPR effect of the NO donor-embedded macromolecular therapeutics. Increasing blood flow in tumor and loosening tight junction of endothelia of tumor vasculature will contribute to the enhance tumor accumulation of the NO donor-embedded macromolecular therapeutics. The enhancement of the EPR effect of the present macromolecular therapeutics was at most two-folds, which is as same as other NO donor-embedded macromolecular therapeutics (polymeric micelle, HSA dimer) reported from other researchers. Thus, this extent of enhancement will be the maximum to be achieved by the NO donor-embedding into macromolecular therapeutics. As described in chapter 1, many molecular systems have been reported to enhance the EPR effect. Some of the molecular systems which are based on the orthogonal mechanism to my system will be able to combine with mine to show the synergy on the enhancement of the EPR effect.
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Acknowledgements
I would like to acknowledge precious experience over the last six years and a great relationship in Katayama laboratory. If I did not meet people who have helped my study in Kyushu University, I could not complete my doctoral thesis.
Firstly, I am deeply grateful to Professor Yoshiki Katayama for his suggestive advice and close assistance during the research. His constant pointer guides my life as a researcher so that my doctoral thesis is fulfilled.
I would like to thank my doctoral thesis committee members; Professor Masaru Tanaka and Professor Yoshiko Miura, for their invaluable advice and questions.
Moreover, special thanks to Associate Professor Takeshi Mori in working through many problems in my research. His valuable advice and helpful assistance give me the power of research accomplishment. I practice a lot of presentation and discussion in my research due to his daily care. My Doctor of Philosophy degree receives his knowledge and encouragement.
I would like to thank the advice of Associate Professor Akihiro Kishimura in polymer science. I also want to thank Jeong-Hun Kang (National Cerebral and Cardiovascular Center) and Associate Professor Tatsuhiro Yamamoto, for their kind advice in various experiments.
I would like to thank Professor Hiroshi Maeda (Kumamoto University) for fruitful discussions. I also would like to thank Professor Toyoshi Iguchi (Kyushu University) for the assistance of blood pressure measurement.
Special thanks to Professor Patrick S. Stayton in accepting me as a visiting scholar at his laboratory at the University of Washington for 9 months. The experience at his laboratory gives me the opportunity to think about my research from different aspects.
I thank this work was financially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. I also thank this work was financially supported by Research Fellowship for Young Scientists (JSPS, 17J04646) and Advanced Graduate Course on Molecular Systems for Devices (Kyushu University).